T cell depletion is a chronic condition complicating many clinical settings including cancer, HIV infection, autoimmune disease. Whereas recovery of depleted myeloid populations is generally rapid and complete, recovery of depleted lymphocytes is generally prolonged and incomplete, resulting in immune dysfunction that can last months or even years. New therapies to enhance lymphocyte recovery would improve morbidity and mortality in a variety of clinical settings. On the other hand, recent studies have demonstrated that the alterations in immune physiology induced by lymphocyte depletion can potentially be used for therapeutic gain in the setting of immunotherapy for cancer. Thus, a better understanding of this immune physiology could lead to new therapies to improve the effectiveness of immunotherapy for cancer. The long-term goals of this project are to improve our understanding of the biology of T cell depletion, to identify the factors limiting full immune reconstitution in humans and to identify new approaches that could be developed to improve immune reconstitution in a variety of clinical settings. We also seek to improve our understanding of the changes in immune physiology induced by lymphopenia, with the goal of developing therapies than can replicate the beneficial effects of lymphopenia while avoiding the adverse effects. Our approach spans small animal models, which allow us to undertake targeted questions, to studies of healthy human cells and studies of patients who have sustained lymphocyte depletion. This bench-to-bedside-to-bench iteration provides insights that increase basic understanding and set the stage for clinical application. Project Summary: 500-7900 characters We have previously demonstrated that humans regenerate T cells via thymic-dependent pathways and via thymic-independent homeostatic peripheral expansion. Thymic dependent pathways are superior in quality due to their capacity to regenerate a broad T cell receptor repertoire however very few humans who sustain T cell depletion have sufficient residual thymic function to efficiently restore T cells using this pathway. Thus, most patients rely on homeostatic peripheral expansion (HPE) to restore T cell populations and HPE typically results in chronically reduced CD4+ T cell numbers, inverted CD4/CD8 ratios and immune dysfunction. Previous paradigms for understanding T cell homeostasis and pathways of immune reconstitution provided no construct for understanding why humans have such differential capacities to restore CD4+ T cells whereas CD8+ populations are readily restored. A major accomplishment of this project during FY2009 was publication of the results of a series of studies that identified the factors limiting CD4+ immune reconstitution via HPE. To summarize, we demonstrated that IL-7 produced by bone marrow derived populations supported efficient CD4+ HPE, whereas production of IL-7 by radioresistant tissues paradoxically diminished CD4+ HPE. Furthermore, elevated IL-7 levels in lymphopenic mice induces downregulation of MHC Class II expression on antigen presenting cell (APCs) populations and diminished IL-7 production by the same APCs. Remarkably, if IL-7 signaling on APCs was inhibited by absence of IL-7 receptor alpha, lack of the common gamma chain receptor or by lack of STAT5, CD4+ HPE was greatly enhanced. Therefore, this work identified an entirely new regulatory axis for controlling CD4+ HPE, which primarily involves IL-7 signaling on APCs. This is paradigm changing since IL-7 signaling on APCs has not previously been shown to be of any real importance, yet this work implicates this axis as a fundamental regulator of CD4+ immune reactivity. This work was published in Nature Immunology in 2009 (Guimond et al, Nat Imm 2009). We are currently extending this work by investigating whether manipulation of IL-7 signaling pathways on APCs effects sensitivity to autoimmunity and/or antitumor effects. Other accomplishment in this arena during FY10 was the publication of a manuscript detailing effects of sunitinib on thymic regeneration and engraftment after stem cell transplantation. We conducted a series of studies that sought to determine whether engraftment after stem cell transplantation could be modulated by interrupting c-kit and/or flt3 ligand signaling by small molecules that are currently FDA approved for the treatment of cancer. We hypothesized that inhibition of these receptor tyrosine kinases should modulate thymopoieisis but no work in this regard had been done. We demonstrated that sunitinib, as a single agent, could substantially augment hematopoietic engraftment following syngeneic stem cell transplantation and could provided an additive effect with irradiation in the allogeneic setting. This report by published in BLOOD in 2010 and provided a starting point for the development of cytotoxin free preparative regimens for gene therapy or stem cell transplantation.